We propose to develop the small Fe-S protein rubredoxin into a paradigm for understanding how a metalloprotein's molecular architecture controls its stability and redox behavior. Comparisons will be made between a mesophilic (Clostridium pasteurianum, optimum growth temperature of 37 degrees C) and a hyperthermophilic (Pyrococcus furiosus, optimum growth temperature of 95 degrees C) rubredoxin to dissect the specific protein determinants that confer the enhanced thermostability of the latter. Mutational analyses and detailed spectroscopic and calorimetric characterization of the mutated proteins will be used to partition the contributions (charge interactions, hydrogen bonding, metal-ligand bonding, etc.) to this stability. Molecular dynamics simulations will be used to predict effects on thermostability of specific mutations; we will test the ability of such simulations to confirm experimentally observed changes in stability. The contribution of the protein in modulating the redox potential of the Fe(S-Cys)4 site will also be explored by mutational analyses. An electrostatic model will be used to predict computationally the effect on redox potential of specific charge mutations and detailed electrochemical measurements (vs. ionic strength and temperature) on mutated proteins will test these predictions. This is expected to allow determination of the temperature dependence of an effective internal protein dielectric constant. Intramolecular electron-transfer kinetics studies will explore the possibility that P. junosus rubredoxin gains its increased stability from stronger or more extensive internal hydrogen bonding. Electronic coupling between the rubredoxin Fe(S-Cys)4 site and attached electron-donor complexes is expected to be sensitive to differences in internal protein architecture and temperature-dependent electron-transfer rate measurements will probe these differences between the P. furiosus and C. pasteurianum proteins. The combination of (a) the comparative nature of this study between two structurally homologous proteins with very different thermostabilities, and (b) the availability of the feedback loop between computational tools and experimental measurements, gives us a unique opportunity to fully characterize the protein determinants of thermostability and the protein tuning of redox potentials and electron-transfer rates. The methods developed in this project will be generally applicable to other metalloprotein systems.